Mab424

The proliferation of the PANC-1 tumor cells was determined by the expression of proliferating cell nuclear antigen (PCNA) using immunohistochemical staining. In brief, tumors were excised from each mouse and weighed at the end of the experiment. Tumor tissues were fixed in buffered formalin for 24 h and then with ethanol for 48 h. Paraffin blocks of tumor tissues were prepared and paraffin sections of tumor tissues were processed for immunohistochemical staining. The sections were incubated with PCNA antibody (MAB424; Millipore Corp.) for 1 h at room temperature. The sections were then incubated with a biotinylated secondary antibody for 30 min followed by incubation with horseradish peroxidase conjugated-avidin solution for 30 min using the Elite ABC kit (PK-6100; Vector Laboratories, Burlingame, CA, USA). PCNA staining in the tumor cells (brown color in nucleus) was examined under a microscope (Nikon Optiphot; Nikon). At least 1,000 cells were counted for each section.

Mouse monoclonal antibody against IL-33 (clone Nessy-1) (#ALX-804-840-C100) was purchased from Enzo. Rabbit polyclonal antibodies against KRT5 (#ab24647) and Ki-67 (#ab15580) were purchased from Abcam (Abcam, Cambridge, MA). Rabbit polyclonal antibody against KRT14 (#PRB-155P) was purchased from Covance (Covance, Princeton, NJ). Rabbit polyclonal antibody against KRT4 (#HPA034881) was purchased from Sigma (Sigma-Aldrich Corp, St. Louis, MO). Rabbit monoclonal antibodies against E-cadherin (#3195), p75 (#8238), and phospho-histone H3 (#3377) were purchased from Cell Signaling (Cell Signaling Technology, MA). Mouse monoclonal antibody against p63 (#sc-8431) was purchased from Santa Cruz (Santa Cruz Biotechnology, TX). Mouse monoclonal antibody against PCNA (#MAB424) was purchased from Millipore (Billerica, MA). Goat polyclonal antibody against IL-33 (#AF3625) was purchased from R&D (R&D Systems, Minneapolis, MN). Donkey anti-goat Alexa Fluor 488 (A11055), anti-rabbit Alexa Fluor 568 (A10042), and anti-mouse Alexa Fluor 647 (A31571) secondary antibodies were purchased from Life Technologies (Carlsbad, CA). Mouse anti-HSP90 (TA500494) and mouse anti-GAPDH (TA310153) primary antibodies were purchased from Origene (Rockville, MD).

Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) was used to determine the proliferation of the PC-3 tumor cells. In brief, paraffin sections of tumor tissues were prepared and processed for immunohistochemical staining. The tumor sections were incubated with a primary PCNA antibody (MAB424, Millipore Corp. Billerica, MA, USA) for 1 h at room temperature. After washed with phosphate buffered saline (PBS), the sections were incubated with a biotinylated secondary antibody for 30 min followed by incubation with horseradish peroxidase conjugated-avidin solution for 30 min using the Elite ABC kit (PK-6100, Vector Laboratories, Burlingame, CA, USA). PCNA staining in tumor cells (brown color in nucleus) were examined under a microscope (Nikon Optiphot, Nikon, Tokyo, Japan). At least 1000 cells were counted for each section.